This invention is concerned with applications of recombinant DNA technology in the field of neurobiology. More particularly, the invention relates to the cloning and expression of DNA coding for excitatory amino acid (EAA) receptors, especially human EAA receptors.
In the mammalian central nervous system (CNS), the transmission of nerve impulses is controlled by the interaction between a neurotransmitter substance released by the xe2x80x9csendingxe2x80x9d neuron which then binds to a surface receptor on the xe2x80x9creceivingxe2x80x9d neuron, to cause excitation thereof. L-glutamate is the most neurotransmitter in the CNS, and mediates the major excitatory pathway is vertebrates. Glutamate is therefore referred to as an excitatory amino acid (EAA) and the receptors which respond to it are variously referred to as glutamate receptors, or more commonly as EAA receptors.
Using tissues isolated from mammalian brain, and various synthetic EAA receptor agonists, knowledge of EAA receptor pharmacology has been refined somewhat. Members of the EAA receptor family are now grouped into three main types based on differential binding to such agonists. One type of EAA receptor, which in addition to glutamate also binds the agonist NMDA (N-methyl-D-aspartate), is referred to as the NMDA type of EAA receptor. Two other glutamate-binding types of EAA receptor, which do not bind NMDA, are named according to their preference for binding with two other EAA receptors agonists, namely AMPA (xcex1-amino-3-hydroxy-5-methyl-isoxazole-4-propionate), and kainate. Particularly, receptors which bind glutamate but not NMDA, and which bind with greater affinity to kainate than to AMPA, are referred to as kainate type EAA receptors. Similarly, those EAA receptors which bind glutamate but not NMDA, and which bind AMPA with greater affinity than kainate are referred to as AMPA type EAA receptors.
The glutamate-binding EAA receptor family is of great physiological and medical importance. Glutamate is involved in many aspects of long-term potentiation (learning and memory), in the development of synaptic plasticity, in epileptic seizures, in neuronal damage caused by ischemia following stroke or other hypoxic events, as well as in other forms of neurodegenerative processes. However, the development of therapeutics which modulate these processes has been very difficult, due to the lack of any homogeneous source of receptor material with which to discover selectively binding drug molecules, which interact specifically at the interface of the EAA receptor. The brain derived tissues currently used to screen candidate drugs are heterogeneous receptor sources, possessing on their surface many receptor types which interfere with studies of the EAA receptor/ligand interface of interest. The search for human therapeutics is further complicated by the limited availability of brain tissue of human origin. It would therefore be desirable to obtain cells that are genetically engineered to produce only the receptor of interest. With cell lines expressing cloned receptor genes, a substrate which is homogeneous for the desired receptor is provided, for drug screening programs.
Non-human cDNAs which appear to encode the kainate-type of receptor have been reported. Egebjerg et al. (Nature 351: 745, 1991) and WO91/06648, each describe the isolation of a cDNA from rat called GluR6 which, although related by sequence to AMPA receptor genes, forms a receptor which is not activated by AMPA but rather by glutamate, quisqualate, and preferentially, kainate. Other kainate binding proteins, which do not readily exhibit ion channel properties when expressed in a homomeric fashion, have also been cloned from frog (Wada et al., Nature 342: 684, 1989), chicken (Gregor et al., Nature 342: 689, 1989; Eshar et al., FEBS Lett. 297: 257, 1992), mouse (Sakimura et al., Neuron 8: 267, 1992) and rat (Werner et al., Nature 351: 742, 1991; Bettler et al., Neuron 8: 257, 1992; Herb et al., Neuron 8: 775, 1992).
There has emerged from these molecular cloning advances a better understanding of the structural features of EAA receptors and their subunits, as they exist in the rat brain. According to the current model of EAA receptor structure, each is heteromeric in structure, consisting of individual membrane-anchored subunits, each having four transmembrane regions, and extracellular domains that dictate ligand binding properties to some extent and contribute to the ion-gating function served by the receptor complex.
In the search for therapeutics useful to treat CNS disorders in humans, it is highly desirable to obtain knowledge of human EAA receptors. A specific understanding of human receptors would provide a means to screen for compounds that react therewith, i.e. to stimulate or inhibit receptor activity, and thus, provides a means to identify compounds having potential therapeutic utility in humans. Non-human mammalian models are not suitable for this purpose despite significant receptor sequence homology as minute sequence differences between species homologues of the same receptor from different species can cause dramatic pharmacological variation (Oksenberg et al., Nature, 360: 161, 1992). It is therefore particularly desirable to provide cloned cDNA encoding human EAA receptors, and cell lines expressing these receptors in a homogeneous fashion, in order to generate a proper screening method for compounds therapeutically useful in humans. These, accordingly, are objects of the present invention.
It is another object of the present invention to provide in isolated form a DNA molecule which codes for a human EAA receptor.
It is another object of the present invention to provide a cell that has been genetically engineered to produce a kainate-binding human EAA receptor.
Other objects of the present invention will be apparent from the following description of the invention.
Polynucleotides coding for a family of EAA receptors which in addition to binding glutamate with an affinity typical of EAA receptors, also exhibit ligand binding properties characteristic of kainate-type EAA receptors, have now been identified and characterized. A representative member of this human EAA receptor family is herein designated human EAA3a. Sequence-related polynucleotides coding for naturally occurring variants of the human EAA3a receptor have also been identified, and constitute additional members of this receptor family, herein referred to as the human EAA3 receptor family.
The present invention thus provides, in one of its aspects, an isolated polynucleotide, consisting either of DNA or of RNA, which codes for a human EAA3 receptor or for a kainate-binding fragment thereof.
In another aspect of the present invention, there is provided a cell that has been genetically engineered to produce a kainate-binding, human EAA receptor belonging to the herein-defined EAA3 family. In related aspects of the present invention, there are provided recombinant DNA constructs and relevant methods useful to create such cells.
In another aspect of the present invention, there is provided a method for evaluating interaction between a test ligand and a human EAA receptor, which comprises the steps of incubating the test ligand with a genetically engineered cell of the present invention, or with a membrane preparation derived therefrom, and then assessing said interaction by determining receptor/ligand binding.